The retinoblastoma protein-associated transcription repressor RBaK interacts with the androgen receptor and enhances its transcriptional activity.

In search of potential androgen receptor coregulators we performed a yeast two-hybrid screening using the androgen receptor ligand-binding domain as bait and a human prostate cDNA library as prey and found that the carboxy-terminal domain of retinoblastoma-associated Krüppel protein (RbaK), a member of the Krüppel zinc finger protein family, interacts in a ligand-dependent way with the ligand-binding domain of the androgen receptor. RBaK was recently identified as a transcriptional regulator that interacts with the retinoblastoma protein and thereby influences E2F regulated transcription. The interaction of RBaK with the androgen receptor was further documented using mammalian two-hybrid experiments, in vitro binding studies and coimmunoprecipitation. Finally, we demonstrated that both RBaK and the retinoblastoma protein coactivate androgen receptor-mediated transcription in cotransfection experiments. In conclusion, our data show that RBaK interacts with the androgen receptor and increases its transcriptional activity. Moreover, the double interaction of RBaK with the retinoblastoma protein and with the androgen receptor provides a novel link between the androgen receptor and the regulation of the cell cycle.

Androgens stimulate lipogenic gene expression in prostate cancer cells by activation of the sterol regulatory element-binding protein cleavage activating protein/sterol regulatory element-binding protein pathway.

Using two independent prostate cancer cell lines (LNCaP and MDA-PCa-2a), we demonstrate that coordinated stimulation of lipogenic gene expression by androgens is a common phenomenon in androgen-responsive prostate tumor lines and involves activation of the sterol regulatory element-binding protein (SREBP) pathway. We show 1) that in both cell lines, androgens stimulate the expression of fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase, two key lipogenic genes representative for the fatty acid and the cholesterol synthesis pathway, respectively; 2) that treatment with androgens results in increased nuclear levels of active SREBP; 3) that the effects of androgens on promoter-reporter constructs derived from both lipogenic genes (fatty acid synthase and hydroxymethylglutaryl-coenzyme A synthase) depend on the presence of intact SREBP-binding sites; and 4) that cotransfection with dominant-negative forms of SREBPs abolishes the effects of androgens. Related to the mechanism underlying androgen activation of the SREBP pathway, we show that in addition to minor effects on SREBP precursor levels, androgens induce a major increase in the expression of sterol regulatory element-binding protein cleavage-activating protein (SCAP), an escort protein that transports SREBPs from their site of synthesis in the endoplasmic reticulum to their site of proteolytical activation in the Golgi. Both time course studies and overexpression experiments showing that increasing levels of SCAP enhance the production of mature SREBP and stimulate lipogenic gene expression support the contention that SCAP plays a pivotal role in the lipogenic effects of androgens in tumor cells.

E2F activity is biphasically regulated by androgens in LNCaP cells.

Androgens exert a peculiar biphasic dose-dependent influence on the proliferation of LNCaP cells, a widely used model to study androgen effects on prostate cancer cells. Low concentrations of androgen stimulate proliferation, but high concentrations inhibit proliferation and induce strong expression of differentiation markers. In order to gain more insight into the molecular mechanisms that underlie these changes we studied the influence of a wide concentration range of the synthetic androgen R1881 on several cell cycle- and differentiation-related parameters. Low concentrations (0.1 nM), known to promote LNCaP cell proliferation, induce an increase of Retinoblastoma protein phosphorylation, accompanied by an increase of E2F-1 protein levels and E2F activity and by increased expression of the E2F-target gene products E2F-1 and cyclin A. High concentrations of R1881 (10 nM) induce strong expression of the differentiation marker prostate-specific antigen. Retinoblastoma protein is largely hypophosphorylated, resulting in low E2F activity and low concentrations of E2F-1 and cyclin A mRNA. Finally, there is a strong increase of p27(KIP1) protein, but not of p27(KIP1) mRNA. These results indicate that the biphasic dose response of LNCaP proliferation to androgen is closely reflected in Rb phosphorylation, E2F activity and p27(KIP1) protein expression.

Apparent coactivation due to interference of expression constructs with nuclear receptor expression.

Transient cotransfection in COS-7 cells, a standard approach to demonstrate coactivation, was used to study the coactivation properties of NuRIP183, a new nuclear receptor interacting protein of 183 kDa, isolated by a yeast two-hybrid screening. Transfection with a NuRIP183 expression construct strongly increased the ligand-dependent response of reporter constructs for several nuclear receptors when compared to transfection with the empty expression vector. A more detailed study, however, revealed major changes in the expression level of the nuclear receptors in cotransfection experiments, indicating that the observed changes in receptor activity were not due to coactivation but to differences in receptor concentration caused by interference from the cotransfected expression constructs with the expression of the receptor. Such interference, which is inversely related to the length of the insert, was observed, not only in COS-7 cells but also in CV-1 and MCF-7 cells, using different transfection techniques (FuGENE-6 and calcium phosphate) and different expression vectors (pSG5, pcDNA1.1 and pIRESneo). These data cast some doubt on coactivation of nuclear receptors based on similar cotransfection experiments without measurement of receptor concentration. Moreover, it is recommended to limit the amounts of (co)transfected expression plasmid and to avoid the use of empty expression plasmid as a control. Finally, one should be aware of similar misleading results in other experimental set-ups based on cotransfection.

Stimulation of tumor-associated fatty acid synthase expression by growth factor activation of the sterol regulatory element-binding protein pathway.

Increased expression of fatty acid synthase (FAS) is observed in a clinically aggressive subset of various common cancers and interference with FAS offers promising opportunities for selective chemotherapeutic intervention. The mechanisms by which FAS expression is (up)-regulated in these tumors remain, however, largely unknown. Recently we demonstrated that in LNCaP prostate cancer cells FAS expression is markedly elevated by androgens via an indirect pathway involving sterol regulatory element-binding proteins (SREBPs). Here, we also show that growth factors such as EGF are able to stimulate FAS mRNA, protein and activity. Several observations also indicate that the effects of EGF on FAS expression are ultimately mediated by SREBPs. EGF stimulates SREBP-1c mRNA expression and induces an increase in mature nuclear SREBP-1. Moreover, in transient transfection studies EGF stimulates the transcriptional activity of a 178 bp FAS promoter fragment harboring a complex SREBP-binding site. Deletion or mutation of this binding site abolishes these effects and ectopic expression of dominant negative SREBP-1 inhibits FAS expression and induction in intact LNCaP cells. Given the frequent dysregulation of growth factor signaling in cancer and the key role of SREBP-1 in lipid homeostasis, growth factor-induced activation of the SREBP pathway is proposed as one of the mechanisms responsible for up-regulation of lipogenic gene expression in a subset of cancer cells.

Selective activation of the fatty acid synthesis pathway in human prostate cancer.

A substantial subset of breast, colorectal, ovarian, endometrial and prostatic cancers displays markedly elevated expression of immunohistochemically detectable fatty acid synthase, a feature that has been associated with poor prognosis and that may be exploited in anti-neoplastic therapy. Here, using an RNA array hybridisation technique complemented by in situ hybridisation, we report that in prostate cancer fatty acid synthase expression is up-regulated at the mRNA level together with other enzymes of the same metabolic pathway. Contrary to the observations that in many cell systems (including androgen-stimulated LNCaP prostate cancer cells) fatty acid and cholesterol metabolism are co-ordinately regulated so as to supply balanced amounts of lipids for membrane biosynthesis, storage or secretion, no changes in the expression of genes involved in cholesterol synthesis were found. These findings point to selective activation of the fatty acid synthesis pathway and suggest a shift in the balance of lipogenic gene expression in a subgroup of prostate cancers.

Progestins and androgens increase expression of Spot 14 in T47-D breast tumor cells.

Enhanced expression of fatty acid synthase and other lipogenic enzymes has been observed in a subset of breast cancers with poor prognosis. This phenomenon has been related to amplification of a gene on chromosome region 11q13 encoding Spot 14, a putative regulator of lipogenic enzyme expression. In this paper we demonstrate that the induction of lipogenesis by progestins and androgens in the breast cancer cell line T47-D is accompanied by a marked increase in the expression of Spot 14. These data corroborate the correlation between Spot 14 expression and increased lipogenesis. Moreover they show that apart from gene amplification there is another steroid-regulated pathway that may enhance Spot 14 expression and lipogenesis in tumor cells.

Androgens down-regulate the expression of the human homologue of paternally expressed gene-3 in the prostatic adenocarcinoma cell line LNCaP.

mRNA differential display polymerase chain reaction analysis was used to screen systematically for novel androgen-regulated genes in the human prostatic adenocarcinoma cell line LNCaP. A 232 bp PCR fragment was found to be consistently down-regulated by the synthetic androgen R1881. Sequencing revealed complete identity with the human homologue of mouse Paternally expressed gene 3 (Peg3), an imprinted gene that plays an important role as a downstream mediator of the effects of tumor necrosis factor (TNF). The down-regulation of Peg3 mRNA by androgens was confirmed by Northern blot hybridization. The effect proved time and dose dependent with maximal repression (3.5-fold) after 24 h of treatment with 10(-8) M R1881. The steroid specificity of Peg3 mRNA regulation reflected the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, supporting the involvement of the androgen receptor in the repression process. Basal expression of Peg3 mRNA was almost completely abolished by the protein synthesis inhibitor cycloheximide. Experiments with Actinomycin D suggested that androgens act at a transcriptional level rather than by changing the stability of Peg3 mRNA. Comparison of the expression of Peg3 mRNA in 50 different human tissues revealed ubiquitous expression, but low levels in the prostate. The highest levels were observed in endocrine tissues such as ovary, placenta, adrenal and pituitary. High levels were also noted in various parts of the brain. No detectable levels of Peg3 mRNA were observed in two other androgen receptor-positive prostate tumor cell lines (MDA PCa-2a and -2b), and in the poorly differentiated and androgen receptor-negative prostate tumor lines PC-3 and DU-145. It is concluded that both androgens and loss of differentiation may affect the expression of Peg3, a mediator of the effects of TNF. Further experiments will be required to explore whether these changes affect the responsiveness of prostate tumor cells to TNF.

The differentiation-related gene 1, Drg1, is markedly upregulated by androgens in LNCaP prostatic adenocarcinoma cells.

A differential display technique was used to identify androgen-regulated genes in LNCaP prostatic adenocarcinoma cells. One of the genes markedly upregulated by androgens proved to be identical to differentiation-related gene 1 (Drg1; also described as RTP, Cap43 and rit42), a gene whose expression has recently been shown to be diminished in colon, breast and prostate tumors. We show that Drg1 is abundantly expressed in the (androgen-exposed) human prostate and that its expression is stimulated some 14-fold in androgen-treated LNCaP cells. The ligand specificity of the induction reflects the altered specificity of the mutated androgen receptor in LNCaP. In androgen receptor negative tumor lines basal expression is slightly higher than in LNCaP but inducibility is absent. These data suggest that Drg1 is a novel marker of androgen-induced differentiation in the human prostate.

Heregulins or neu differentiation factors and the interactions between peritubular myoid cells and Sertoli cells.

Interactions between (mesenchymal) peritubular myoid cells and (epithelial) Sertoli cells play an important role in the control of Sertoli cell function and spermatogenesis. The factors involved, however, have only partially been identified. Heregulins or neu differentiation factors (NDFs) mediate mesenchymal-epithelial interactions in a variety of tissues, but their role in the testis has not been investigated. Here we demonstrate that recombinant human heregulin-alpha (Her-alpha) and Her-beta stimulate transferrin and androgen-binding protein production by cultured rat Sertoli cells up to 2.5-fold. These effects are more pronounced than those of previously identified growth factors active in this assay, such as insulin-like growth factor I and basic fibroblast growth factor. Combination with these factors results in additive effects and in marked morphological changes in the Sertoli cell cultures, including formation of tubule-like structures. Stimulation of androgen-binding protein secretion is paralleled by increased levels of the corresponding messenger RNA. This parallelism was less consistent for transferrin. Semiquantitative RT-PCR indicates that the expression of NDF-alpha and NDF-beta is more pronounced in peritubular cells than in Leydig or Sertoli cells. Conversely, the main receptors for heregulins/NDFs, HER-3 and HER-4, are predominantly expressed in Sertoli cells. A displacement assay confirms the presence of high-affinity binding sites for [125I]Her-beta on intact Sertoli cells and reveals parallel displacement curves for Her-beta, Her-alpha, and concentrated peritubular cell-conditioned medium (PTCM; estimated ED50 values: 1 ng/ml, 50 ng/ml, and 130 microg protein/ml, respectively), indicating that peritubular cells secrete one or more factors able to compete for heregulin receptors. It is concluded that heregulins/NDFs may play a role in mesenchymal-epithelial interactions in the testis. Estimates of the concentrations of heregulins in PTCM, however, make it unlikely that they contribute significantly to the effects observed with low concentrations of PTCM and ascribed to the putative mediator PModS (peritubular factor that modulates Sertoli cell function). Further investigations will be required to define the exact role of heregulins in the testis.

Identification of diazepam-binding Inhibitor/Acyl-CoA-binding protein as a sterol regulatory element-binding protein-responsive gene.

Diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP), a highly conserved 10-kDa polypeptide, has been implicated in various physiological processes including gamma-aminobutyric acid type A receptor binding, acyl-CoA binding and transport, steroidogenesis, and peptide hormone release. Both in LNCaP prostate cancer cells and 3T3-L1 preadipocytes, the expression of DBI/ACBP is stimulated under conditions that promote lipogenesis (treatment with androgens and insulin, respectively) and that involve the activation of sterol regulatory element-binding proteins (SREBPs). Accordingly, we investigated whether DBI/ACBP expression is under the direct control of SREBPs. Analysis of the human and rat DBI/ACBP promoter revealed the presence of a conserved sterol regulatory element (SRE)-like sequence. Gel shift analysis confirmed that this sequence is able to bind SREBPs. In support of the functionality of SREBP binding, coexpression of SREBP-1a with a DBI/ACBP promoter-reporter gene resulted in a 50-fold increase in transcriptional activity in LNCaP cells. Disruption of the SRE decreased basal expression and abolished SREBP-1a-induced transcriptional activation. In agreement with the requirement of a co-regulator for SREBP function, transcriptional activation by SREBP-1a overexpression was severely diminished when a neighboring NF-Y site was mutated. Cholesterol depletion or androgen treatment, conditions that activate SREBP function in LNCaP cells, led to an increase in DBI/ACBP mRNA expression and SRE-dependent transcriptional activation. These findings indicate that the promoter for DBI/ACBP contains a functional SRE that allows DBI/ACBP to be coregulated with other genes involved in lipid metabolism.

Expression of cystatin-related protein and of the C3-component of prostatic-binding protein during postnatal development in the rat ventral prostate and lacrimal gland.

The expression of cystatin-related protein (CRP) and of the C3-component of prostatic-binding protein (PBP) during postnatal development of the rat was studied by Northern blotting, dot blot and in situ hybridisation, and by radioimmunoassay or immunoblotting. In intact male rats, very little or no PBP-C3 could be detected in the prostate at 10 days, but at 20 days there was already strong expression. By in situ hybridisation, the first expression of C3 mRNA was observed at 13 days in the prostate and at 22 days in the lacrimal gland. For CRP, this occurred at 16 and 22 days, respectively. Neither CRP nor C3 was expressed in prepubertal male rats castrated at day 1 or day 10 or in female rats. Androgen treatment of intact male animals did not advance the expression of both mRNAs in the prostate, but did so in the lacrimal gland with first expression of C3 at 19 instead of 22 days and of CRP at 13 instead of 22 days. Identical values were obtained in female rats. Androgen treatment of castrated adult male rats resulted in a more rapid and homogeneous secondary induction. Positive immunostaining for the androgen receptor (AR) was observed in the lacrimal gland at 7 days, but its concentration, estimated by immunoblotting, was still low at 10 days. Maximal levels, reached at 30 days, were markedly higher in male than in female rats. In conclusion, CRP and C3 are induced by androgens in prepubertal rats. The time point of induction, however, is probably determined by other tissue and differentiation-dependent factors in addition to androgens and the AR.

Primary rat lacrimal cells undergo acinar-like morphogenesis on reconstituted basement membrane and express secretory component under androgen stimulation.

Single cells or small cell clusters, isolated from the rat lacrimal gland, were incubated on reconstituted basement membrane (matrigel) in a well-defined serum-free medium. During the first days of culture, cells reassociated and reorganized in structures resembling acini. These multicellular structures, maintained in culture for 2 weeks, consisted of well-polarized cuboidal cells surrounding a central lumen and exhibiting apically located microvilli. Myoepithelial cells were observed at the periphery of the acinar structures. Both in the native lacrimal and in the cultured aggregates, epithelial cells displayed strong immunoreactivity for cytokeratin 8, while myoepithelial cells were immunoreactive for vimentin and alpha-smooth muscle isoactin. These data indicate that the cultured aggregates closely mimic the in vivo architecture of lacrimal glands both by morphology and immunohistochemistry. We further demonstrated the presence of an intact androgen receptor and the ability of the cultured aggregates to respond to androgens with increased secretion of the secretory component. Comparable androgen responses were observed in lacrimal gland cultures of 5-week-old male and female rats. In conclusion, we report a morphologically and functionally differentiated culture system of primary rat lacrimal cells, in which androgen-regulated gene expression was observed. This culture model provides a unique experimental paradigm for studying the effects of hormones, cytokines, and growth factors on the morphogenesis, growth, and functional differentiation of lacrimal glands.

Identification of the phosphatidic acid phosphatase type 2a isozyme as an androgen-regulated gene in the human prostatic adenocarcinoma cell line LNCaP.

Differential display was used to identify novel androgen-regulated genes in the human prostatic adenocarcinoma cell line LNCaP. A 322-base pair cDNA fragment that was consistently induced by the synthetic androgen R1881 revealed 100% homology with the human phosphatidic acid phosphatase type 2a isozyme very recently reported by Kai et al. (PAP-2a; Kai., M., Wada, I., Imai, S.-i., Sakane, F., and Kanoh, H. (1997) J. Biol. Chem. 272, 24572-24578). The fragment was used to clone the corresponding cDNA from a human prostate library. The deduced amino acid sequence confirmed the identity with human PAP-2a. The inducibility of PAP-2a mRNA by androgens was confirmed by Northern blot hybridization. The effect was time- and dose-dependent with a maximal stimulation (4-fold) after 24 h of treatment with 10(-9) M R1881. The steroid specificity of PAP-2a mRNA regulation was found to be in agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, supporting the involvement of the androgen receptor in the induction process. Furthermore, low basal levels of PAP-2a mRNA and absence of androgen inducibility were observed in the poorly differentiated and androgen receptor-negative cell lines PC-3 and DU-145. Induction of PAP-2a mRNA was not affected by the protein synthesis inhibitor cycloheximide and was accompanied by a marked increase in PAP-2 activity as measured by the conversion of phosphatidic acid into diacylglycerol in membrane fractions of LNCaP. Comparison of the expression of PAP-2a mRNA in 50 different human tissues revealed ubiquitous expression. The highest levels, however, were observed in the prostate. Since PAP-2 plays a pivotal role in the control of signal transduction by lipid mediators such as phosphatidate, lysophosphatidate, and ceramide-1-phosphate, the ability of androgens to stimulate the expression and activity of this enzyme in prostatic cells may provide an important opportunity for cross-talk between signaling pathways involving lipid mediators and androgens.

Occult ectopic ACTH secretion due to recurrent lung carcinoid: long-term control of hypercortisolism by continuous subcutaneous infusion of octreotide.

We report on a woman with clinical Cushing's syndrome confirmed by biochemical data. The Cushing's syndrome was shown to be ACTH dependent and inferior petrosal sinus sampling pointed to an ectopic source. After resection of a lung carcinoid a well documented remission of Cushing's syndrome was obtained. At recurrence of Cushing's syndrome 18 months later the ACTH source could not be located despite extensive diagnostic procedures. Clinical and biochemical control of hypercortisolism was achieved by continuous subcutaneous infusion of octreotide. During a brief interruption of treatment recurrence of clinical and biochemical signs and symptoms of Cushing's syndrome were demonstrated. We conclude that in this case of occult ectopic ACTH secretion, presumably due to recurrent lung carcinoid, continuous subcutaneous infusion therapy with octreotide resulted in clinical and metabolic control of Cushing's syndrome for 8 years. In addition excellent tumour growth control was achieved as repeated searches for tumour recurrence or metastasis remained negative.

Coordinate regulation of lipogenic gene expression by androgens: evidence for a cascade mechanism involving sterol regulatory element binding proteins.

To gain more insight into the molecular mechanisms by which androgens stimulate lipogenesis and induce a marked accumulation of neutral lipids in the human prostate cancer cell line LNCaP, we studied their impact on the expression of lipogenic enzymes. Northern blot analysis of the steady-state mRNA levels of seven different lipogenic enzymes revealed that androgens coordinately stimulate the expression of enzymes belonging to the two major lipogenic pathways: fatty acid synthesis and cholesterol synthesis. In view of the important role of the recently characterized sterol regulatory element binding proteins (SREBPs) in the coordinate induction of lipogenic genes, we examined whether the observed effects of androgens on lipogenic gene expression are mediated by these transcription factors. Our findings indicate that androgens stimulate the expression of SREBP transcripts and precursor proteins and enhance the nuclear content of the mature active form of the transcription factor. Moreover, by using the fatty acid synthase gene as an experimental paradigm we demonstrate that the presence of an SREBP-binding site is essential for its regulation by androgens. These data support the hypothesis that SREBPs are involved in the coordinate regulation of lipogenic gene expression by androgens and provide evidence for the existence of a cascade mechanism of androgen-regulated gene expression.

LNCaP prostatic adenocarcinoma cells derived from low and high passage numbers display divergent responses not only to androgens but also to retinoids.

In the present paper, two strains of LNCaP cells derived from the same source (American Type Culture Collection), but studied either at a low passage number (LP) or at a high passage number (HP), were compared in their response to R1881 (a synthetic androgen), all-trans-retinoic acid (atRA), and 1alpha,25-dihydroxycholecalciferol (VD3). [3H]Thymidine incorporation and epidermal growth factor receptor (EGF-R) binding were measured as parameters related to the proliferative response of the cells. The secretion of prostate-specific antigen (PSA) and the mRNA expression of PSA, prostatic acid phosphatase (PAP), and diazepam-binding inhibitor (DBI) were used as parameters reflecting differentiated function. Marked differences were noted in the response of LP and HP cells to androgens. [3H]Thymidine incorporation displayed a bell-shaped dose-response curve in both strains. The amplitude of the response, however, was much higher in HP cells and growth inhibition at high levels of R1881 was only observed in LP cells. On the contrary, androgen induction of PSA secretion and PSA mRNA expression, as well as the expression of PAP was much more pronounced in LP cells, whereas DBI expression was not altered according to passage number. LP cells and HP cells also displayed striking differences in their response to atRA. An up to 6-fold stimulation of [3H]thymidine incorporation was observed in LP cells, whereas in HP cells the only significant effect was growth inhibition. VD3, on the contrary, inhibited [3H]thymidine incorporation to a comparable degree in LP and HP cells. Only marginal effects of atRA and VD3 were observed on PSA secretion. In both LP and HP cells EGF-R levels were increased by androgens and to a slight extent also by atRA and VD3. It is concluded that LP and HP LNCaP cells display markedly divergent responses not only to androgens but also to atRA. The proliferative rather than antiproliferative effects of atRA in some strains of LNCaP should caution against the uncontrolled use of these agents, or of drugs affecting their metabolism, in patients with prostate cancer.

Identification of a functional androgen-response element in the exon 1-coding sequence of the cystatin-related protein gene crp2.

Two hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2, +40/+63). It displays an androgen response element-like sequence motif 5'-AGAAGAaaaTGTACA-3' and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites, ARBSu/crp1 and ARBSu/crp2, occur 5' upstream of the transcription start point and are located at an identical position (-142/ -120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (-273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5'-TGTACA-3' sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs in T-47D cells. However, the induction measured is less than the one observed with crp2F (-273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androgen responsiveness.

Androgens stimulate fatty acid synthase in the human prostate cancer cell line LNCaP.

In addition to modulation of cell proliferation and stimulation of prostate-specific antigen secretion, one of the most striking effects of androgens on the human prostate cancer cell line LNCaP is the accumulation of neutral lipids. These lipids are synthesized de novo, suggesting that LNCaP cells express all enzymes required for endogenous lipogenesis and that the expression and/or activity of some of these enzymes is affected by androgens. One of the key enzymes involved in lipogenesis is fatty acid synthase (FAS), a potential prognostic enzyme and therapeutic target that is found to be frequently overexpressed in a variety of cancers including prostate cancer. Here, using Northern blot analysis, the gene encoding FAS is shown to be abundantly expressed in LNCaP cells and in two other prostate cancer cell lines tested (PC-3 and DU-145). In LNCaP cells, androgen treatment (10(-8) M R1881) causes a 3-4-fold increase in FAS mRNA levels. Concomitantly with the increase in FAS gene expression, androgens induce a 10-12-fold stimulation of FAS activity. Effects are dose- and time-dependent and follow courses similar to those of the androgen induction of lipid accumulation. In support of the involvement of the androgen receptor, steroid specificity of regulation of FAS activity is in agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells. Stimulation of FAS activity is inhibited by the antiandrogen Casodex (bicalutamide) and is absent in the androgen receptor-negative cell lines PC-3 and DU-145. Taken together, these data demonstrate that androgens, mediated by the androgen receptor, stimulate the expression and activity of FAS and suggest that stimulation of FAS activity represents at least part of the mechanism by which androgens induce the accumulation of neutral lipids in LNCaP cells.